As shown previously, glucagon responsiveness was selectively lost in MDCK cells, a dog kidney cell line, after transformation by Harvey murine sarcoma virus. This responsiveness can be restored to the transformed MDCK cells by culturing the cells in the presence of various inducers, including butyrate, prostaglandie E1 and Ro 20-1724, an inhibitor of cyclic AMP-phosphodiesterase. Now we have shown that 8-Br-cyclic AMP also induces glucagon sensitivity. However, the effect of cyclic AMP is biphasic; at high concentrations, cyclic AMP induces only poorly, in fact, it inhibits the effect of other inducers when added simultaneously. All inducers, with the exception of butyrate, work more effectively in the defined medium, i.e., in the absence of serum. This observation led to our discovery of a differentiation inhibitor. We have now partially purified this factor from horse serum. At a protein concentration of 1 Mug/ml, this factor completely inhibited the induction of glucagon sensitivity by prostaglandins but not by butyrate. Our current effort is directed toward defining the biochemical events responsible for cyclic AMP involvement in the induction process and the role of differentiation inhibitor. Using a photoaffinity label, (125I)-azido-phenylamidinoglucagon, glucagon receptors in liver plasma membranes was specifically labeled and subject to purification by SDS-PAGE and isoelectrofocusing. Glucagon receptors purified as such exhibit a Mr of 55000 and an isoelectric point of 5.8. Using this material as antigen, immunized rabbits produced antisera strongly immunoreactive with MDCK cells containing glucagon receptors. In contrast, these antisera reacted only poorly with a transformed MDCK cell line lacking glucagon receptors. However, these antisera did not inhibit binding of (125I)-glucagon to liver plasma membranes nor did it block activation of cyclic AMP production by glucagon. We are currently characterizing these antibodies to assure that they indeed recognize glucagon receptors.